Increased pathogen exposure of a marine apex predator over three decades.

Environmental changes associated with global warming create new opportunities for pathogen and parasite transmission in Arctic wildlife. As an apex predator ranging over large, remote areas, changes in pathogens and parasites in polar bears are a useful indicator of changing transmission dynamics in Arctic ecosystems. We examined prevalence and risk factors associated with exposure to parasites and viral and bacterial pathogens in Chukchi Sea polar bears. Serum antibodies to six pathogens were detected and prevalence increased between 1987-1994 and 2008-2017 for five: Toxoplasma gondii, Neospora caninum, Francisella tularensis, Brucella abortus/suis, and canine distemper virus. Although bears have increased summer land use, this behavior was not associated with increased exposure. Higher prevalence of F. tularensis, Coxiella burnetii, and B. abortus/suis antibodies in females compared to males, however, could be associated with terrestrial denning. Exposure was related to diet for several pathogens indicating increased exposure in the food web. Elevated white blood cell counts suggest a possible immune response to some pathogens. Given that polar bears face multiple stressors in association with climate change and are a subsistence food, further work is warranted to screen for signs of disease.

Giardia and Cryptosporidium in resident wildlife species in Arctic Alaska.

Giardia and Cryptosporidium are zoonotic protozoan parasites that can infect humans and other taxa, including wildlife, often causing gastrointestinal illness. Both have been identified as One Health priorities in the Arctic, where climate change is expected to influence the distribution of many wildlife and zoonotic diseases, but little is known about their prevalence in local wildlife. To help fill information gaps, we collected fecal samples from four wildlife species that occur seasonally on the northern Alaska coastline or in nearshore marine waters-Arctic fox (Vulpes lagopus), polar bear (Ursus maritimus), Pacific walrus (Odobenus rosmarus divergens), and caribou (Rangifer tarandus)-and used immunofluorescence assays to screen for Giardia cysts and Cryptosporidium oocysts. We detected Giardia cysts in 18.3% and Cryptosporidium oocysts in 16.5% of Arctic foxes (n = 109), suggesting that foxes may be potentially important hosts in this region. We also detected Giardia cysts in a single polar bear (12.5%; n = 8), which to our knowledge represents the first such report for this species. Neither parasite was detected in walruses or caribou.

A SEROLOGIC SURVEY OF FRANCISELLA TULARENSIS EXPOSURE IN WILDLIFE ON THE ARCTIC COASTAL PLAIN OF ALASKA, USA.

Tularemia is an infectious zoonotic disease caused by one of several subspecies of Francisella tularensis bacteria. Infections by F. tularensis are common throughout the northern hemisphere and have been detected in more than 250 wildlife species. In Alaska, US, where the pathogen was first identified in 1938, studies have identified F. tularensis antibodies in a diverse suite of taxa, including insects, birds, and mammals. However, few such investigations have been conducted recently and knowledge about the current distribution and disease ecology of F. tularensis is limited, particularly in Arctic Alaska, an area undergoing rapid environmental changes from climate warming. To help address these information gaps and provide insights about patterns of exposure among wildlife, we assessed the seroprevalence of F. tularensis antibodies in mammals and tundra-nesting geese from the Arctic Coastal Plain of Alaska, 2014-17. With a commercially available slide agglutination test, we detected antibodies in 14.7% of all individuals sampled (n=722), with titers ranging from 1:20 to 1:320. We detected significant differences in seroprevalence between family groups, with Canidae (foxes, Vulpes spp.) and Sciuridae (Arctic ground squirrel, Spermophilus parryii) having the highest seroprevalence at 21.5% and 33.3%, respectively. Mean seroprevalence for Ursidae (polar bears, Ursus maritimus) was 13.3%, whereas Cervidae (caribou, Rangifer tarandus) had comparatively low seroprevalence at 6.5%. Antibodies were detected in all Anatidae species sampled, with Black Brant (Branta bernicla nigricans) having the highest seroprevalence at 13.6%. The detection of F. tularensis antibodies across multiple taxa from the Arctic Coastal Plain and its nearshore marine region provides evidence of exposure to this pathogen throughout the region and highlights the need for renewed surveillance in Alaska.

Paralytic shellfish toxins associated with Arctic Tern mortalities in Alaska.

Harmful algal blooms produce biotoxins that can injure or kill fish, wildlife, and humans. These blooms occur naturally but have intensified in many locations globally due to recent climatic changes, including ocean warming. Such changes are especially pronounced in northern regions, where the effects of paralytic shellfish toxins (PSTs) on marine wildlife are of growing concern. In Alaska, seabird mortality events have increased in frequency, magnitude, and duration since 2015 alongside anomalously high ocean temperatures. Although starvation has been implicated as the apparent cause of death in many of these die-offs, saxitoxin (STX) and other PSTs have been identified as possible contributing factors. Here, we describe a mortality event at a nesting colony of Arctic Terns (Sterna paradisaea) near Juneau, Alaska in 2019 and report elevated concentrations of PSTs in bird, forage fish, and mussel samples. Concentrations of STX and other PSTs in tern tissues (2.5-51.2 µg 100g-1 STX-equivalents [STX-eq]) were of similar magnitude to those reported from other PST-induced bird die-offs. We documented high PST concentrations in blue mussels (>11,000 µg 100g-1 STX-eq; Mytilus edulis spp.) collected from nearby beaches, as well as in forage fish (up to 494 µg 100g-1 STX-eq) retrieved from Arctic Tern nests, thereby providing direct evidence of PST exposure via the terns' prey. At maximum concentrations measured in this study, a single 5 g Pacific Sand Lance (Ammodytes personatus) could exceed the median lethal STX dose (LD50) currently estimated for birds, offering strong support for PSTs as a likely source of tern mortality. In addition to describing this localized bird mortality event, we used existing energetics data from adult and nestling Arctic Terns to calculate estimated cumulative daily PST exposure based on ecologically relevant concentrations in forage fish. Our estimates revealed potentially lethal levels of PST exposure even at relatively low (≤30 ug 100g-1 STX-eq) toxin concentrations in prey. These findings suggest that PSTs present a significant hazard to Arctic Terns and other northern seabirds and should be included in future investigations of avian mortality events as well as assessments of population health.

Acute oral toxicity and tissue residues of saxitoxin in the mallard (Anas platyrhynchos).

Since 2014, widespread, annual mortality events involving multiple species of seabirds have occurred in the Gulf of Alaska, Bering Sea, and Chukchi Sea. Among these die-offs, emaciation was a common finding with starvation often identified as the cause of death. However, saxitoxin (STX) was detected in many carcasses, indicating exposure of these seabirds to STX in the marine environment. Few data are available that describe the effects of STX in birds, thus presenting challenges for determining its contributions to specific mortality events. To address these knowledge gaps, we conducted an acute oral toxicity trial in mallards (Anas platyrhynchos), a common laboratory avian model, using an up-and-down method to estimate the median lethal dose (LD50) for STX. Using an enzyme-linked immunosorbent assay (ELISA), we tested select tissues from all birds and feces from those individuals that survived initial dosing. Samples with an ELISA result that exceeded approximately 10 µg 100 g-1 STX and randomly selected ELISA negative samples were further tested by high-performance liquid chromatography (HPLC). Tissues collected from mallards were also examined grossly at necropsy and then later by microscopy to identify lesions attributable to STX. The estimated LD50 was 167 µg kg-1 (95% CI = 69-275 µg kg-1). Saxitoxin was detected in fecal samples of all mallards tested for up to 48 h after dosing and at the end of the sampling period (7 d) in three birds. In those individuals that died or were euthanized <2 h after dosing, STX was readily detected throughout the gastrointestinal tract but only infrequently in heart, kidney, liver, lung, and breast muscle. No gross or microscopic lesions were observed that could be attributable to STX exposure. Given its acute toxicity, limited detectability, and frequent occurrence in the Alaska marine environment, additional research on STX in seabirds is warranted.

Investigation of Algal Toxins in a Multispecies Seabird Die-Off in the Bering and Chukchi Seas.

Between 2014 and 2017, widespread seabird mortality events were documented annually in the Bering and Chukchi seas, concurrent with dramatic reductions of sea ice, warmer than average ocean temperatures, and rapid shifts in marine ecosystems. Among other changes in the marine environment, harmful algal blooms (HABs) that produce the neurotoxins saxitoxin (STX) and domoic acid (DA) have been identified as a growing concern in this region. Although STX and DA have been documented in Alaska (US) for decades, current projections suggest that the incidence of HABs is likely to increase with climate warming and may pose a threat to marine birds and other wildlife. In 2017, a multispecies die-off consisting of primarily Northern Fulmars (Fulmarus glacialis) and Short-tailed Shearwaters (Ardenna tenuirostris) occurred in the Bering and Chukchi seas. To evaluate whether algal toxins may have contributed to bird mortality, we tested carcasses collected from multiple locations in western and northern Alaska for STX and DA. We did not detect DA in any samples, but STX was present in 60% of all individuals tested and in 88% of Northern Fulmars. Toxin concentrations in Northern Fulmars were within the range of those reported from other STX-induced bird die-offs, suggesting that STX may have contributed to mortalities. However, direct neurotoxic action by STX could not be confirmed and starvation appeared to be the proximate cause of death among birds examined in this study.

POECIVIRUS IS PRESENT IN INDIVIDUALS WITH BEAK DEFORMITIES IN SEVEN SPECIES OF NORTH AMERICAN BIRDS.

Avian keratin disorder (AKD), a disease of unknown etiology characterized by debilitating beak overgrowth, has increasingly affected wild bird populations since the 1990s. A novel picornavirus, poecivirus, is closely correlated with disease status in Black-capped Chickadees (Poecile atricapillus) in Alaska, US. However, our knowledge of the relationship between poecivirus and beak deformities in other species and other geographic areas remains limited. The growing geographic scope and number of species affected by AKD-like beak deformities require a better understanding of the causative agent to evaluate the population-level impacts of this epizootic. Here, we tested eight individuals from six avian species with AKD-consistent deformities for the presence of poecivirus: Mew Gull (Larus canus), Hairy Woodpecker (Picoides villosus), Black-billed Magpie (Pica hudsonia), American Crow (Corvus brachyrhynchos), Red-breasted Nuthatch (Sitta canadensis), and Blackpoll Warbler (Setophaga striata). The birds were sampled in Alaska and Maine (1999-2016). We used targeted PCR followed by Sanger sequencing to test for the presence of poecivirus in each specimen and to obtain viral genome sequence from virus-positive host individuals. We detected poecivirus in all individuals tested, but not in negative controls (water and tissue samples). Furthermore, we used unbiased metagenomic sequencing to test for the presence of other pathogens in six of these specimens (Hairy Woodpecker, two American Crows, two Red-breasted Nuthatches, Blackpoll Warbler). This analysis yielded additional viral sequences from several specimens, including the complete coding region of poecivirus from one Red-breasted Nuthatch, which we confirmed via targeted PCR followed by Sanger sequencing. This study demonstrates that poecivirus is present in individuals with AKD-consistent deformities from six avian species other than Black-capped Chickadee. While further investigation will be required to explore whether there exists a causal link between this virus and AKD, this study demonstrates that poecivirus is not geographically restricted to Alaska, but rather occurs elsewhere in North America.

Algal toxins in Alaskan seabirds: Evaluating the role of saxitoxin and domoic acid in a large-scale die-off of Common Murres.

Elevated seawater temperatures are linked to the development of harmful algal blooms (HABs), which pose a growing threat to marine birds and other wildlife. During late 2015 and early 2016, a massive die-off of Common Murres (Uria aalge; hereafter, murres) was observed in the Gulf of Alaska coincident with a strong marine heat wave. Previous studies have documented illness and death among seabirds resulting from exposure to the HAB neurotoxins saxitoxin (STX) and domoic acid (DA). Given the unusual mortality event, corresponding warm water anomalies, and recent detection of STX and DA throughout coastal Alaskan waters, HABs were identified as a possible factor of concern. To evaluate whether algal toxins may have contributed to murre deaths, we tested for STX and DA in a suite of tissues obtained from beach-cast murre carcasses associated with the die-off as well as from apparently healthy murres and Black-legged Kittiwakes (Rissa tridactyla; hereafter, kittiwakes) sampled in the preceding and following summers. We also tested forage fish and marine invertebrates collected in the Gulf of Alaska in 2015-2017 to evaluate potential sources of HAB toxin exposure for seabirds. Saxitoxin was present in multiple tissue types of both die-off (36.4 %) and healthy (41.7 %) murres and healthy kittiwakes (54.2 %). Among birds, we detected the highest concentrations of STX in liver tissues (range 1.4-10.8 µg 100 g-1) of die-off murres. Saxitoxin was relatively common in forage fish (20.3 %) and invertebrates (53.8 %). No established toxicity limits currently exist for seabirds, but concentrations of STX in birds and forage fish in our study were lower than values reported from most other bird die-offs in which STX intoxication was causally linked. We detected low concentrations of DA in a single bird sample and in 33.3 % of invertebrates and 4.0 % of forage fish samples. Although these results do not support the hypothesis that acute exposure to STX or DA was a primary factor in the 2015-2016 mortality event, additional information about the sensitivity of murres to these toxins is needed before we can discount their potential role in the die-off. The widespread occurrence of STX in seabirds, forage fish, and invertebrates in the Gulf of Alaska indicates that algal toxins should be considered in future assessments of seabird health, especially given the potential for greater occurrence of HABs in the future.

Cryptic and extensive hybridization between ancient lineages of American crows.

Most species and therefore most hybrid zones have historically been defined using phenotypic characters. However, both speciation and hybridization can occur with negligible morphological differentiation. Recently developed genomic tools provide the means to better understand cryptic speciation and hybridization. The Northwestern Crow (Corvus caurinus) and American Crow (Corvus brachyrhynchos) are continuously distributed sister taxa that lack reliable traditional characters for identification. In this first population genomic study of Northwestern and American crows, we use genomic SNPs (nuDNA) and mtDNA to investigate the degree of genetic differentiation between these crows and the extent to which they may hybridize. Our results indicate that American and Northwestern crows have distinct evolutionary histories, supported by two nuDNA ancestry clusters and two 1.1%-divergent mtDNA clades dating to the late Pleistocene, when glacial advances may have isolated crow populations in separate refugia. We document extensive hybridization, with geographic overlap of mtDNA clades and admixture of nuDNA across >900 km of western Washington and western British Columbia. This broad hybrid zone consists of late-generation hybrids and backcrosses, but not recent (e.g., F1) hybrids. Nuclear DNA and mtDNA clines had concordant widths and were both centred in southwestern British Columbia, farther north than previously postulated. Overall, our results suggest a history of reticulate evolution in American and Northwestern crows, perhaps due to recurring neutral expansion(s) from Pleistocene glacial refugia followed by lineage fusion(s). However, we do not rule out a contributing role for more recent potential drivers of hybridization, such as expansion into human-modified habitats.

Prevalence and diversity of avian blood parasites in a resident northern passerine.

BACKGROUND: Climate-related changes are expected to influence the prevalence and distribution of vector-borne haemosporidian parasites at northern latitudes, although baseline information about resident birds is still lacking. In this study, we investigated prevalence and genetic diversity of Plasmodium, Haemoproteus, and Leucocytozoon parasites infecting the northwestern crow (Corvus caurinus), a non-migratory passerine with unique life-history characteristics. This species occupies both intertidal and forested habitats and is subject to high prevalence of avian keratin disorder (AKD), a disease that causes gross beak deformities. Investigation of avian blood parasites in northwestern crows at sites broadly distributed across coastal Alaska provided an opportunity to evaluate specific host factors related to parasite infection status and assess geographical patterns of prevalence. RESULTS: We used molecular methods to screen for haemosporidian parasites in northwestern crows and estimated genus-specific parasite prevalence with occupancy modeling that accounts for imperfect detection of parasite infection. We observed considerable geographical and annual variation in prevalence of Plasmodium, Haemoproteus, and Leucocytozoon, but these patterns were not correlated with indices of local climatic conditions. Our models also did not provide support for relationships between the probability of parasite infection and body condition or the occurrence of co-infections with other parasite genera or clinical signs of AKD. In our phylogenetic analyses, we identified multiple lineages of each parasite genus, with Leucocytozoon showing greater diversity than Plasmodium or Haemoproteus. CONCLUSIONS: Results from this study expand our knowledge about the prevalence and diversity of avian blood parasites in northern resident birds as well as corvids worldwide. We detected all three genera of avian haemosporidians in northwestern crows in Alaska, although only Leucocytozoon occurred at all sites in both years. Given the strong geographical and annual variation in parasite prevalence and apparent lack of correlation with climatic variables, it appears that there are other key factors responsible for driving transmission dynamics in this region. Thus, caution is warranted when using standard climatic or geographical attributes in a predictive framework. Our phylogenetic results demonstrate lower host specificity for some lineages of Leucocytozoon than is typically reported and provide insights about genetic diversity of local haemosporidian parasites in Alaska.

Evidence of Culiseta mosquitoes as vectors for Plasmodium parasites in Alaska.

Mosquito vectors play a crucial role in the distribution of avian Plasmodium parasites worldwide. At northern latitudes, where climate warming is most pronounced, there are questions about possible changes in the abundance and distribution of Plasmodium parasites, their vectors, and their impacts to avian hosts. To better understand the transmission of Plasmodium among local birds and to gather baseline data on potential vectors, we sampled a total of 3,909 mosquitoes from three locations in south-central Alaska during the summer of 2016. We screened mosquitoes for the presence of Plasmodium parasites using molecular techniques and estimated Plasmodium infection rates per 1,000 mosquitoes using maximum likelihood methods. We found low estimated infection rates across all mosquitoes (1.28 per 1,000), with significantly higher rates in Culiseta mosquitoes (7.91 per 1,000) than in Aedes mosquitoes (0.57 per 1,000). We detected Plasmodium in a single head/thorax sample of Culiseta, indicating potential for transmission of these parasites by mosquitoes of this genus. Plasmodium parasite DNA isolated from mosquitoes showed a 100% identity match to the BT7 Plasmodium lineage that has been detected in numerous avian species worldwide. Additionally, microscopic analysis of blood smears collected from black-capped chickadees (Poecile atricapillus) at the same locations revealed infection by parasites preliminarily identified as Plasmodium circumflexum. Results from our study provide the first information on Plasmodium infection rates in Alaskan mosquitoes and evidence that Culiseta species may play a role in the transmission and maintenance of Plasmodium parasites in this region.

Serologic Evidence for Influenza A Virus Exposure in Three Loon Species Breeding in Alaska, USA.

Limited information exists about exposure to influenza A viruses (IAVs) in many wild waterbird species, including loons. We analyzed serum samples from breeding adult Pacific (Gavia pacifica), Red-throated (Gavia stellata), and Yellow-billed (Gavia adamsii) loons sampled at three locations along the coast of Alaska, US from 2008 to 2017 to gain a better understanding of the potential role loons play in IAV ecology. We screened loon sera for IAV antibodies using three tests-blocking enzyme-linked immunosorbent assay (bELISA), agar gel immunodiffusion (AGID), and hemagglutination inhibition (HI)-and examined patterns in seroprevalence among species and sampling locations. We found evidence of IAV infection in all loon species and at all breeding locations, although concordance was imperfect among serological tests. Diagnostic tests yielded seroprevalence estimates of 24% (42/172) with bELISA, 8% (5/60) with AGID, and 6% (4/70) with HI. The IAV subtypes to which loon sera reacted using HI were consistent with those detected in waterfowl and gulls at other locations in Alaska, suggesting that loons may be exposed to IAV maintained in sympatric waterbirds. Our study provided evidence that loons inhabiting Alaska were exposed to IAV. However, given imperfect concordance among serologic tests, and relatively low seroprevalence as compared to other avian taxa exposed to IAV in Alaska, they make poor IAV surveillance targets.

Avian keratin disorder of Alaska black-capped chickadees is associated with Poecivirus infection.

BACKGROUND: Avian keratin disorder (AKD) is an epizootic of debilitating beak deformities, first documented in black-capped chickadees (Poecile atricapillus) in Alaska during the late 1990s. Similar deformities have now been recorded in dozens of species of birds across multiple continents. Despite this, the etiology of AKD has remained elusive, making it difficult to assess the impacts of this disease on wild populations. We previously identified an association between infection with a novel picornavirus, Poecivirus, and AKD in a small cohort of black-capped chickadees. METHODS: To test if the association between Poecivirus and AKD holds in a larger study population, we used targeted PCR followed by Sanger sequencing to screen 124 symptomatic and asymptomatic black-capped chickadees for Poecivirus infection. We further compared the efficacy of multiple non-terminal field sampling methods (buccal swabs, cloacal swabs, fecal samples, and blood samples) for Poecivirus screening. Finally, we used both in situ hybridization and a strand-specific expression assay to localize Poecivirus to beak tissue of AKD-positive individuals and to determine if virus is actively replicating in beak tissue. RESULTS: Poecivirus was detected in 28/28 (100%) individuals with AKD, but only 9/96 (9.4%) asymptomatic individuals with apparently normal beaks (p < 0.0001). We found that cloacal swabs are the most sensitive of these sample types for detecting Poecivirus in birds with AKD, but that buccal swabs should be combined with cloacal swabs in evaluating the infection status of asymptomatic birds. Finally, we used both in situ hybridization and a strand-specific expression assay to localize Poecivirus to beak tissue of AKD-positive individuals and to provide evidence of active viral replication. CONCLUSION: The data presented here show a strong, statistically significant relationship between Poecivirus infection and AKD, and provide evidence that Poecivirus is indeed an avian virus, infecting and actively replicating in beak tissue of AKD-affected BCCH. Taken together, these data corroborate and extend the evidence for a potential causal association between Poecivirus and AKD in the black-capped chickadee. Poecivirus continues to warrant further investigation as a candidate agent of AKD.

Morphological and molecular characterization of Sarcocystis arctica-like sarcocysts from the Arctic fox (Vulpes lagopus) from Alaska, USA.

The muscles of herbivores commonly harbor sarcocysts of parasites belonging to species in the genus Sarcocystis, but such muscle parasites are rare in carnivores. Here, we report Sarcocystis arctica-like sarcocysts in muscles of Arctic foxes (Vulpes lagopus) from Alaska, USA, for the first time. The tongues of 56 foxes were examined for Sarcocystis infection using several methods. Sarcocystis bradyzoites were detected in pepsin digests of 13 (23.2%), and sarcocysts were found in histological sections stained with hematoxylin and eosin (HE) of 9 (16.0%). By light microscopy, sarcocysts were up to 4 mm long and up to 245 µm wide. In HE-stained sections, the sarcocyst wall appeared smooth and up to 1.5 µm thick without visible protrusions. By transmission electron microscopy, the sarcocyst wall had a wavy parasitophorous vacuolar membrane (pvm) folded as pleomorphic villar protrusions (vp), sometimes with anastomoses of villar tips. The vp and the ground substance (gs) layer were smooth and without microtubules. The gs was up to 2.0 µm thick. The total width of the wall including vp and the gs was up to 4.0 µm. The vp were up to 3.0 µm long and most closely resembled "type 9c." All sarcocysts were mature and contained numerous 8.1 × 2.1 µm sized bradyzoites. Molecular characterization (at 18S rDNA, 28S rDNA, ITS-1, and cox1) showed the highest affinity for S. arctica of the Arctic fox (V. lagopus) from Norway. In the present investigation, we provide evidence that sarcocysts are common in tongues of Alaskan Arctic foxes suggesting that these carnivores are serving as intermediate hosts, and we also provide ultrastructure of S. arctica from the Arctic fox for the first time.

Haemosporidian parasite infections in grouse and ptarmigan: Prevalence and genetic diversity of blood parasites in resident Alaskan birds.

Projections related to future climate warming indicate the potential for an increase in the distribution and prevalence of blood parasites in northern regions. However, baseline data are lacking for resident avian host species in Alaska. Grouse and ptarmigan occupy a diverse range of habitat types throughout the northern hemisphere and are among the most well-known and important native game birds in North America. Information regarding the prevalence and diversity of haemosporidian parasites in tetraonid species is limited, with few recent studies and an almost complete lack of genetic data. To better understand the genetic diversity of haemosporidian parasites in Alaskan tetraonids and to determine current patterns of geographic range and host specificity, we used molecular methods to screen 459 tissue samples collected from grouse and ptarmigan species across multiple regions of Alaska for infection by Leucocytozoon, Haemoproteus, and Plasmodium blood parasites. Infections were detected in 342 individuals, with overall apparent prevalence of 53% for Leucocytozoon, 21% for Haemoproteus, and 9% for Plasmodium. Parasite prevalence varied by region, with different patterns observed between species groups (grouse versus ptarmigan). Leucocytozoon was more common in ptarmigan, whereas Haemoproteus was more common in grouse. We detected Plasmodium infections in grouse only. Analysis of haemosporidian mitochondrial DNA cytochrome b sequences revealed 23 unique parasite haplotypes, several of which were identical to lineages previously detected in other avian hosts. Phylogenetic analysis showed close relationships between haplotypes from our study and those identified in Alaskan waterfowl for Haemoproteus and Plasmodium parasites. In contrast, Leucocytozoon lineages were structured strongly by host family. Our results provide some of the first genetic data for haemosporidians in grouse and ptarmigan species, and provide an initial baseline on the prevalence and diversity of blood parasites in a group of northern host species.

Novel Picornavirus Associated with Avian Keratin Disorder in Alaskan Birds.

UNLABELLED: Avian keratin disorder (AKD), characterized by debilitating overgrowth of the avian beak, was first documented in black-capped chickadees (Poecile atricapillus) in Alaska. Subsequently, similar deformities have appeared in numerous species across continents. Despite the widespread distribution of this emerging pathology, the cause of AKD remains elusive. As a result, it is unknown whether suspected cases of AKD in the afflicted species are causally linked, and the impacts of this pathology at the population and community levels are difficult to evaluate. We applied unbiased, metagenomic next-generation sequencing to search for candidate pathogens in birds affected with AKD. We identified and sequenced the complete coding region of a novel picornavirus, which we are calling poecivirus. Subsequent screening of 19 AKD-affected black-capped chickadees and 9 control individuals for the presence of poecivirus revealed that 19/19 (100%) AKD-affected individuals were positive, while only 2/9 (22%) control individuals were infected with poecivirus. Two northwestern crows (Corvus caurinus) and two red-breasted nuthatches (Sitta canadensis) with AKD-consistent pathology also tested positive for poecivirus. We suggest that poecivirus is a candidate etiological agent of AKD. IMPORTANCE: Avian keratin disorder (AKD) is an increasingly common disease of wild birds. This disease, characterized by beak overgrowth, was first described in the late 1990s and has been spreading rapidly both geographically and in terms of host species affected. AKD decreases host fitness and can be fatal. However, the cause of the disease has remained elusive, and its impact on host populations is poorly understood. We found a novel and divergent picornavirus in 19/19 AKD-affected black-capped chickadees that we examined but in only 2/9 control cases. We also found this virus in 4 individuals of 2 other passerine species that exhibited symptoms consistent with AKD. Our data suggest that this novel picornavirus warrants further investigation as the causative agent of AKD.

Blood Serum Chemistry of Wild Alaskan Black-capped Chickadees ( Poecile atricapillus ) with Avian Keratin Disorder.

We measured serum chemistries in wild Black-capped Chickadees ( Poecile atricapillus ) from Alaska to test for potential differences associated with beak deformities characteristic of avian keratin disorder. Lower uric acid in affected birds was the only difference detected between groups, although sample sizes were small. This difference could be associated with fasting or malnutrition in birds with beak deformities, but it is challenging to interpret its biologic significance without reference values. Black-capped Chickadees had high levels of aspartate aminotransferase, lactate dehydrogenase, and creatine kinase relative to reference values for companion birds. However, all serum chemistry parameters from our study were within the range of values reported from other apparently healthy wild-caught birds.

Elements in Whole Blood of Northwestern Crows ( Corvus caurinus ) in Alaska, USA: No Evidence for an Association with Beak Deformities.

A recent outbreak of beak deformities among resident birds in Alaska, US, has raised concern about environmental contamination as a possible underlying factor. We measured whole blood concentrations of 30 essential and nonessential elements to determine whether any were associated with beak deformities in Northwestern Crows ( Corvus caurinus ). We tested for differences between 1) adults with versus those without beak deformities and 2) unaffected adults versus juveniles. Crows with beak deformities had slightly higher levels of barium, molybdenum, and vanadium (all P<0.05), but concentrations were generally low and within the range of values reported from other apparently healthy wild birds. Concentrations of several elements, including selenium, were higher in birds without versus birds with beak deformities (all P<0.05), a difference that may be explained in part by compromised foraging ability associated with the deformities. Adult crows had higher concentrations of cadmium, silicon, and zinc than juveniles (all P<0.05), although differences were relatively small and values were similar to those from other wild birds. Our results suggest that neither selenium nor other tested elements are likely to be causing beak deformities in Alaskan crows. We also provide the first data on elemental concentrations in Northwestern Crows. Levels of selenium far exceeded those typically found in passerine birds and were similar to those in marine-associated waterfowl, suggesting that background levels should be interpreted relative to a species' environment.

Avian malaria in a boreal resident species: long-term temporal variability, and increased prevalence in birds with avian keratin disorder.

The prevalence of vector-borne parasitic diseases is widely influenced by biological and ecological factors. Environmental conditions such as temperature and precipitation can have a marked effect on haemosporidian parasites (Plasmodium spp.) that cause malaria and those that cause other malaria-like diseases in birds. However, there have been few long-term studies monitoring haemosporidian infections in birds in northern latitudes, where weather conditions can be highly variable and the effects of climate change are becoming more pronounced. We used molecular methods to screen more than 2,000 blood samples collected from black-capped chickadees (Poecile atricapillus), a resident passerine bird. Samples were collected over a 10 year period, mostly during the non-breeding season, at seven sites in Alaska, USA. We tested for associations between Plasmodium prevalence and local environmental conditions including temperature, precipitation, site, year and season. We also evaluated the relationship between parasite prevalence and individual host factors of age, sex and presence or absence of avian keratin disorder. This disease, which causes accelerated keratin growth in the beak, provided a natural study system in which to test the interaction between disease state and malaria prevalence. Prevalence of Plasmodium infection varied by year, site, age and individual disease status but there was no support for an effect of sex or seasonal period. Significantly, birds with avian keratin disorder were 2.6 times more likely to be infected by Plasmodium than birds without the disorder. Interannual variation in the prevalence of Plasmodium infection at different sites was positively correlated with summer temperatures at the local but not statewide scale. Sequence analysis of the parasite cytochrome b gene revealed a single Plasmodium spp. lineage, P43. Our results demonstrate associations between prevalence of avian malaria and a variety of biological and ecological factors. These results also provide important baseline data that will be informative for predicting future changes in Plasmodium prevalence in the subarctic.

Isolation of a Complete Circular Virus Genome Sequence from an Alaskan Black-Capped Chickadee (Poecile atricapillus) Gastrointestinal Tract Sample.

We report here the genome sequence of a circular virus isolated from samples of an Alaskan black-capped chickadee (Poecile atricapillus) gastrointestinal tract. The genome is 2,152 bp in length and is most similar (30 to 44.5% amino acid identity) to the genome sequences of other single-stranded DNA (ssDNA) circular viruses belonging to the gemycircularvirus group.